Topoisomerase enzymes control the supercoiled state of DNA and are involved in biological processes such as DNA replication and transcription. Vaccinia virus topoisomerase (VT) is a protypical type Ib enzyme closely related to human topoisomerase I (anti-cancer target). Specific peptide inhibitors of VT activity will be isolated. These inhibitors will aid in understanding the catalytic mechanism and the nature of topoisomerase inhibition. The inhibitor peptides will be identified biochemically for their ability to inhibit VT binding, cleavage and religation of their DNA substrate and tested for anti-viral activity, targeting VT using an in vivo model. Inhibitors will be further characterized using mutant VT enzymes (with defined point mutations), highlighting residues involved in enzyme activity that are affected by the inhibitory peptide. Furthermore, regions of VT that interact with the peptides will be identified by peptide-enzyme crosslinking. Finally, the inhibitory effects resulting in DNA conformational changes will be identified by DNA footprinting.